Field of the Invention
The present invention is in the field of analytical electrophoresis systems and methods. The invention can include high resolution and highly sensitive Isotachophoresis (ITP) and capillary electrophoresis (CE) assays.
Description of the Related Art
Electrophoresis is generally the movement of charged molecules in an electric field. Analytical methods based on electrophoresis have found broad utility, especially in the fields of protein and nucleic acid analyses. Samples having charged analyte molecules of interest can be placed in a selective media, such as size exclusion media, ion exchange media, or media having a pH gradient, where they can differentially migrate for high resolution from other sample molecules. The separated molecules can be detected for identification and quantitation.
Capillary and microfluidic scale electrophoretic separations are particularly popular for analyses requiring low sample volumes or high throughput. For example, chips of plastic or glass substrate can be fabricated with microscale loading channels, separation channels and detection channels. Samples can be transferred from microwell plates through a robotically manipulated sample collection tube to the loading channel. An electric potential can induce movement of sample constituents through selective media in the separation channel for sequential detection as the constituents elute into the detection channel from the separation channel. The microscale dimensions of the assay system can provide rapid analyses using microscale, or nanoscale, sample volumes. However, resolution or sensitivity may not be adequate for complex samples or dilute samples.
One approach to enhancing the resolution and sensitivity of capillary electrophoresis (CE) methods has been to pre-resolve and pre-concentrate the sample using Isotachophoresis (ITP) before CE separations. In ITP, the sample is loaded into a channel between a leading electrolyte (LE) having an electrophoretic mobility greater than the sample and a trailing electrolyte (TE) having electrophoretic mobility less than the sample. Under the influence of an electric field, analytes of interest can migrate through the sample bolus to accumulate at the interface with the LE and/or TE solutions. In this way, the analytes of interest can be separated from certain other constituents of the sample and concentrate to more detectable levels. Samples can thus be concentrated and desalted to provide improved injection material for further capillary electrophoresis separations resulting in highly sensitive detections with high resolution. For example, in “Tandem Isotachophoresis-Zone Electrophoresis via Base-Mediated Destacking for Increased Detection Sensitivity in Microfluidic Systems”, by Vreeland, et al., Anal. Chem. (2003) ASAP Article, sample concentrated by ITP is further resolved and detected by capillary zone electrophoresis (CZE). In Vreeland, the sample is subjected to ITP between a TE and an LE having electrophoretic mobilities controlled by the pH of Tris buffers. While ITP concentration of analytes progresses, hydroxyl ions (—OH) are formed by hydrolysis at the cathode end of the separation channel. Migration of the hydroxyl ions through the separation channel eventually neutralizes the Tris buffers to remove the mobility differences between the LE and TE solutions. The Tris neutralization converts the ITP separation media into a CZE separation media. The analytes can than be separated with higher sensitivity and resolution than for standard CZE of the same sample due to the effective sample volume reduction and concentration of analytes resulting from the ITP assay step. The Vreeland method is limited to pH based ITP of compatible samples, can be time consuming due to the neutralization step, and can be inconsistent due to variations in buffer preparation or hydroxyl ion generation.
In another scheme to combine ITP with CE, analytes of interest migrate in ITP mode until they reach an intersection with a CE separation channel before switching the electric field to the separation channel for capillary electrophoresis separation of the analytes. For example, in “Sample Pre-concentration by Isotachophoresis in Microfluidic Devices”, by Wainright, et al., J. Chromat. A979 (2002), pp. 69-80, samples are pre-concentrated in a ITP channel until they reach an intersection with a CE channel. The intersection is monitored microscopically by a photomultiplier tube (PMT) receiving light through a confocal lens focused on the intersection. Analytes entering the intersection can be detected, e.g., by fluorescence or light absorption, and the electric field manually switched to inject the analytes into the CE channel. Problems exist, however, in that the manual switching can be inconsistent, some analytes may not be detectable using a PMT, and PMT detection at the microscale can be cumbersome and expensive.
In view of the above, a need exists for increased sensitivity, consistency, and resolution of capillary and microscale electrophoresis methods. It would be desirable to have systems that can automatically and consistently switch between electrophoretic modes. The present invention provides these and other features that will be apparent upon review of the following.